Trade-offs with stability modulate innate and mutationally acquired drug resistance in bacterial dihydrofolate reductase enzymes

Abstract

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In the present study, we show that trimethoprim (TMP)-resistant alleles of dihydrofolate reductase from (EcDHFR) harboring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild-type, making them prone to aggregation and proteolysis. This destabilization is associated with a lower expression level, resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis, we show that perturbation of critical stabilizing hydrophobic interactions in wild-type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial dihydrofolate reductases (DHFRs). Mutational and computational analyses in EcDHFR and in DHFR enzymes from and demonstrate that natural variation at this site and its interacting hydrophobic residues modulates TMP resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to TMP. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.

Highly Contingent Phenotypes of Lon Protease Deficiency in Escherichia coli upon Antibiotic Challenge

“…This tussle between chaperones and proteases was found to influence the availability of DHFR in bacterial cells and hence to modulate organismal fitness (28). The findings of the present study relate that idea to stability-function trade-offs occurring during the evolution of drug resistance in bacteria, now an established phenomenon (29,31,(46)(47)(48). I propose that destabilization of DHFR due to mutations that confer trimethoprim resistance results in its degradation, presumably by Lon itself, which provides a simple mechanism explaining why certain mutations are able to confer trimethoprim resistance in only Londeficient E. coli.…”

“…4D). Previous work demonstrated that the Trp30Gly mutation destabilizes DHFR (31). I reasoned, therefore, that the Trp30Gly mutation may render DHFR more prone to proteolysis in Lon-expressing bacteria.…”

“…Three of these, namely, Phe137, Phe153, and Ile155 (Fig. 6C), interact with Trp30 in a hydrophobic tetrad and play a role in stabilizing the DHFR fold (31). Since several proteases, including Lon, recognize exposed hydrophobic residues as signals for proteolysis (38), I asked whether mutations at Phe137, Phe153, and Ile155 would also destabilize DHFR in Lon-expressing bacteria.…”

“…Clinical resistance to this antibiotic is mediated primarily by acquisition of drug-resistant, plasmid-encoded DHFR (30). However, genomic resistance to trimethoprim does evolve rapidly in laboratory strains of E. coli and primarily maps to a few mutational hot spots within endogenous DHFR, encoded by the folA gene (31)(32)(33). This has made genomic trimethoprim resistance in E. coli an attractive model to investigate the evolution of drug resistance (29,(31)(32)(33).…”

“…However, genomic resistance to trimethoprim does evolve rapidly in laboratory strains of E. coli and primarily maps to a few mutational hot spots within endogenous DHFR, encoded by the folA gene (31)(32)(33). This has made genomic trimethoprim resistance in E. coli an attractive model to investigate the evolution of drug resistance (29,(31)(32)(33). I report that the phenotypic effects of Lon deficiency varied qualitatively and quantitatively depending on drug concentration, being beneficial at low drug concentrations but detrimental in the presence of high drug concentrations.…”